The present invention relates to oligopeptide derivatives of the formula (I) 
in which
R1 is
(a) a hydrogen atom, a C2-8-alkanoyl group optionally having an amino group in the xcfx89 position, a phenyl-C2-4-alkanoyl group whose phenyl radical is optionally substituted in the p position by an amino group; or
(b) a cyclohexylcarbonyl group which is optionally substituted in the 4 position by an aminomethyl radical, a benzoyl group which is optionally substituted in the o or p position by methyl, amino or halogen, a C1-8-alkoxycarbonyl group, a benzyloxycarbonyl group which is optionally substituted in the p position by methoxy, methyl or chlorine; or
(c) a radical of the formula xe2x80x94SO2xe2x80x94R5 where R5 can be a C1-6-alkyl radical, an optionally substituted aryl or heteroaryl radical or a radical of a bicyclic terpene derivative; or
(d) a group of the formula xe2x80x94COxe2x80x94CH(R6)xe2x80x94NHxe2x80x94R7, where R6 is hydrogen, a C1-6-alkyl radical, a 1- or 2-hydroxyethyl radical, a methylmercaptoethyl radical, an aminobutyl radical, a guanidinopropyl radical, a carboxy-C1-4-alkyl radical, a carboxamido-C1-4-alkyl radical, a phenyl-C1-4-alkyl radical whose phenyl radical is optionally substituted by OH, halogen, C1-4-alkyl or methoxy, or a cyclohexyl or cyclohexylmethyl radical whose ring is optionally substituted by OH, halogen, C1-4-alkyl or methoxy, or a nitrogen-containing heteroaryl-C1-4-alkyl radical with 3 to 8 carbon atoms in the heterocyclic system, where the group xe2x80x94COxe2x80x94CH(R6)xe2x80x94NHxe2x80x94R7 may be racemic or have the D or L configuration, and R7 can be a group of type (a), (b) or (c); or
(e) a group of the formula 
where R7 has the above meaning, m can be 1 or 2, and one of the methylene groups can be substituted by hydroxyl, carboxyl, C1-4-alkyl or aryl-C1-4-alkyl;
R2 is hydrogen, C1-6-alkyl, C1-2-hydroxyalkyl, C1-4-alkoxy-C1-6-alkyl, benzyloxy-C1-2-alkyl, an xcfx89-carboxy-C1-3-alkyl radical, an xcfx89-C1-4-alkoxycarbonyl-C1-3-alkyl radical, an xcfx89-benzyloxycarbonyl-C1-3-alkyl radical or a cyclohexyl, cyclohexylmethyl, 4-hydroxycyclohexylmethyl, phenyl, benzyl, 4-hyroxybenzyl or imidazolyl-4-methyl radical;
R3 is
(a) hydrogen or C1-4-alkyl and R3xe2x80x2 is hydrogen; or
(b) together with R3xe2x80x2 a tri- or tetramethylene group, where one of the methylene groups can be substituted by hydroxyl, carboxyl, C1-4-alkyl or aryl-C1-4-alkyl; and
R4 is
(a) an aniline residue of the formula 
where R8 can be hydroxyl or amino and R9 can be hydrogen, halogen, amino, nitro, C1-4-alkyl, C1-4-alkoxy or C1-4-alkanoyl; or
(b) a quinoline residue of the formula 
where one of R10, R11 and R12 is an xe2x80x94NH group via which the quinoline residue is linked to the Arg residue, a second one can be hydroxyl or amino, and the third one can be hydrogen, hydroxyl or amino;
and the salts thereof.
These oligopeptide derivatives and the salts are novel. They are cleaved by enzymes of the peptide hydrolase enzyme class (E.C. 3.4.), in particular proteinases (E.C. 3.4.21-99) and inhibitors thereof, of the blood coagulation system, of the fibrinolytic system and of complement, in particular of thrombin. They thus serve as substrates for the quantitative and qualitative determination of the abovementioned enzymes, in particular thrombin, in complex sample liquids, in particular capillary blood.
The present invention relates to novel oligopeptide derivatives of the formula (I) and salts thereof, to the preparation of these oligopeptide derivatives and salts, and to a method for the quantitative determination of a protease or antiprotease, in particular of the blood coagulation system, of the fibrinolytic system or of complement, in particular thrombin, which is carried out by means of the novel oligopeptide derivatives or salts thereof.
The term xe2x80x9calkylxe2x80x9d used in this description designates, on its own or in combinations such as xe2x80x9chydroxyalkylxe2x80x9d or xe2x80x9cbenzyloxyalkylxe2x80x9d, straight-chain or branched saturated hydrocarbon radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and the like. The term xe2x80x9calkoxyxe2x80x9d designates an alkyl radical within the meaning of the above definition of xe2x80x9calkylxe2x80x9d, which is linked via an oxygen bridge. The term xe2x80x9calkanoylxe2x80x9d designates the acyl radical of an alkylcarboxylic acid which may be straight-chain or branched, such as acetyl, propionyl and the like. The term xe2x80x9carylxe2x80x9d designates the radical of an aromatic hydrocarbon and comprises radicals such as phenyl, naphthyl and the like. The term xe2x80x9cheteroarylxe2x80x9d designates the radical of an aromatic heterocyclic system such as imidazolyl, indolyl, quinolinyl, isoquinolinyl and the like.
If R1 in formula (I) is a radical of the formula xe2x80x94SO2R5, then R5 can be, for example, methyl, isopropyl, phenyl, tert-butylphenyl, 4-methylphenyl, 2,4,6-trimethylphenyl, 2,4,6-triisopropylphenyl, 4-methoxy-2,3,6-trimethylphenyl, anthraquinoyl, 1- or 2-naphtyl, quinolyl or isoquinolyl or a radical derived from camphor. If, in formula (I), R1 is a group of the formula xe2x80x94COxe2x80x94CH(R6)xe2x80x94NHxe2x80x94R7 and R6 is heteroarylalkyl, then R6 can be, for example, imidazolylmethyl or indolylmethyl.
If the N-terminal amino acid in the molecule carries a protective group, then this protective group is expediently tert-butoxycarbonyl (xe2x80x9cBocxe2x80x9d), p-toluenesulfonyl (xe2x80x9cTosxe2x80x9d), tert-butylphenylsulfonyl (xe2x80x9ct-Bupsxe2x80x9d), methylsulfonyl (xe2x80x9cMesxe2x80x9d), naphthylsulfonyl (xe2x80x9cNapsxe2x80x9d), benzoyl (xe2x80x9cBzoxe2x80x9d), benzyloxycarbonyl (xe2x80x9cZxe2x80x9d), isopropylsulfonyl or camphorsulfonyl.
If R4 is an aniline residue, then expediently R8 is hydroxyl in the o- or p position and R9 is halogen in the m position or R8 is hydroxyl in the o, m or p position and R9 is nitro or C1-4-alkanoyl in the m or p position. The radical R4 is preferably derived from 2-amino-4-nitrophenol, 4-amino-2-nitrophenol, 4-amino-3-nitrophenol, 2-amino-5-nitrophenol, 2,4-diaminotoluene, 2,4-diaminophenol, 4-amino-m-cresol, 2,5-diaminoanisole, 4-nitro-o-phenylenediamine, 2-amino-4-chlorophenol, 4-amino-2-chlorophenol, 4-amino-3-chlorophenol, 4-fluoro-2-aminophenol, 2-fluoro-4-aminophenol, 5-fluoro-2-aminophenol, 5-amino-8-hydroxyquinoline or 2-amino-8-hydroxyquinoline.
The oligopeptide derivatives of the formula (I) contain L-arginine (xe2x80x9cArgxe2x80x9d) as C-terminal amino acid. Further amino acids which may be present in the molecule are, for example, 2-aminobutyric acid, alanine, 3-cyclohexylalanine, 2-cyclohexylglycine, phenylalanine, pipecolic acid, proline and valine, it being possibe for these amino acids to be in the L, D or DL form, and glycine; preference is given in this connection to L-alanine (xe2x80x9cAlaxe2x80x9d), L-2-aminobutyric acid (xe2x80x9cAbuxe2x80x9d), D-3-cyclohexylalanine (xe2x80x9cD-Chaxe2x80x9d), D-2-cyclohexylglycine (xe2x80x9cD-Chgxe2x80x9d), glycine (xe2x80x9cGlyxe2x80x9d) and L-proline (xe2x80x9cProxe2x80x9d).
Representative examples of oligopeptide derivatives of the formula (I) are:
H-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
Boc-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
Tos-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
t-Bups-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
Mes-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
Naps-2-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
Z-(D)-Chg-Gly-Arg 3-chloro-4-hydroxyanilide;
H-(D)-Cha-Gly-Arg 3-chloro-4-hydroxyanilide;
Boc-(D)-Cha-Gly-Arg 3-chloro-4-hydroxyanilide;
H-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
Boc-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
Tos-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
t-Bups-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
Mes-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
isopropylsulfonyl-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
Naps-2-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
(xe2x88x92)-camphorsulfonyl-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
H-(D)-Cha-Pro-Arg 3-chloro-4-hydroxyanilide;
Boc-(D)-Cha-Pro-Arg 3-chloro-4-hydroxyanilide;
H-(D)-Cha-Ala-Arg 3-chloro-4-hydroxyanilide;
Boc-(D)-Cha-Ala-Arg 3-chloro-4-hydroxyanilide;
Boc-(D)-Cha-Abu-Arg 3-chloro-4-hydroxyanilide;
Z-Gly-Pro-Arg 2-chloro-4-hydroxyanilide;
Z-Gly-Pro-Arg 5-chloro-2-hydroxyanilide;
Z-Gly-Pro- Arg 8-hydroxyquinolin-5-ylamide;
Boc-(D)-Chg-Gly-Pro-Arg 3-chloro-4-hydroxyanilide;
H-(D)-Chg-Gly-Pro-Arg 3-chloro-4-hydroxyanilide; and
t-Bups-(D)-Chg-Gly-Pro-Arg 3-chloro-4-hydroxyanilide.
In the bloodstream numerous proteinases reach their site of action in the body. This underlines the importance of knowledge of the enzymic activity for understanding a wide variety of pathological states. Derangements of the balance between enzyme and inhibitor are responsible for the onset of an illness. It is therefore of inestimable value to be able to determine the enzymic activity present.
Blood or plasma are, however, not easy to handle as analysis media because of their properties (viscosity, absorption of visible light). In most cases, enzyme assays are carried out by photometric methods. There are limits to this especially in the case of blood, plasma or serum.
Photometric detection methods usually require clear sample solutions because the chromogenic properties of the synthetic substrates used and the chromophores thereof otherwise cannot have an effect. This often gives rise to preceding purification steps in order to obtain a measurable solution (compare DE 3616496 A1 and DE 19549117 A1).
Measurements of the enzymes of the blood coagulation system are, for example, d desired and carried out in patients with heart valve replacement or after myocardial infarctions. Patients of these types are subjected to a therapy with oral anticoagulants. The intention therewith is to prevent further thrombo-embolic events. For this purpose, the patients are treated at regular intervals with medicaments from the class of coumarin derivatives. However, this therapy requires monitoring for optimal dosage. The parameter of choice in order r to be able to derive an optimal dosage is determination of the Quick prothrombin time (PT).
The oligopeptide derivatives according g to the present invention make it possible to determine the prothrombin time using a substrate which generates an electrochemical signal. It is additionally possible to generate a sufficiently good signal from capillary blood.
European patent EP 0018002 B1 describes an electrochemical method for the determination of proteases of the blood coagulation systems, in particular of antithrombin, in citrated plasma using amperogenic substrates. Unfortunately, inaccuracies of measurement are possible in this elegant method for direct determination of thrombin or trypsin, because the addition of a solubilizer (DMSO) to the reaction medium is necessary to keep the amperogenic substrate dissolved in the reaction medium (J. M. Nigretto et al., Thrombosis Research 20, 299-306, 1980; Thrombosis Research 22, 303-308). The addition of a cosolvent may lead to an inaccuracy in the measurement of thrombin for practical use, for example screenings in the complex system of blood plasma or blood, for example through inducing coagulation, compromising an enzyme in the cascade, local substrate precipitation effects or the like.
The oligopeptide derivatives of the present invention now make possible a novel, direct amperogenic thrombin determination method which is better adapted to practice than previously disclosed determination methods. In this method, the increase in a water-soluble amperogenic compound resulting from the hydrolysis of the substrate by the enzyme is measured. This involves cleavage of a peptide residue whose C-terminal peptide residue is L-arginine, and of an electroactive residue which is connected by an amide linkage to the L-arginine, by the enzyme. The electroactive residue is then electrochemically oxidized or reduced, the change in the current is measured, and the latter is proportional to the concentration of the amperogenic residue formed during the enzymatic hydrolysis.
The method of the invention makes quantitative determination of a protease of antiprotease possible, in particular of the blood coagulation system, of the fibrinolytic system or of complement, in particular thrombin; it is characterized in that the enzyme is contacted in an aqueous or organic medium with an oligopeptide derivative of the formula (I) or a salt thereof, and the electroactive amine of the formula Hxe2x80x94R4, in which R4 has the above meaning, which is cleaved off by the enzyme to be determined and which can be electrochemically oxidized or reduced, is determined by amperometry.
The amperometry is expediently carried out using an apparatus with a potentiostat and a measuring cell with two or three electrodes, a measuring electrode made of steel or a noble metal, such as platinum or gold, a reference electrode and/or an auxiliary electrode, where the oligopeptide derivative of the formula (I) or salt thereof and possible water-soluble additions such as Ca++ salts, phospholipids or thromboplastin reagents is or are applied to the electrodes.
The change in the concentration of the electroactive amine of the formula R4xe2x80x94H in which R4 has the above meaning can be determined in the measuring cell through the measured oxidation or reduction current.
In a preferred embodiment of the determination method of the invention, a direct adhesion of these water-soluble substrates and of possible water-soluble additions such as, for example Ca++, phospholipids, Innovin(copyright) (from DADE-Behring) on a conducting surface made of steel or noble metal is possible, with the necessary electrodes being separated by an isolated bar. After addition of media which contain thrombin or which has been generated beforehand, for example in the blood or blood plasma with activators, for example Ca++, phospholipids, thromboplastin reagents or the like, the substrate is completely dissolved so that, through the liberation of the amperogenic amino compound, a direct determination of the current, proportional to the concentration of the electroactive amino compound, and thus the determination of the enzyme concentration in the medium, is possible.
It is surprising that, in contrast to EP 0 565 665, a mixture of 0-35% of L-xcex1-phosphatidylcholine (PC) and 65-100% of L-xcex1-phosphatidyl-L-serine (PS) is preferably used as phospholipids.
A particularly preferred mixing ratio of the phospholipids PC:PS is 65 to 75% PS and 25 to 35% PC.
The determination method of the invention can be used to determine thrombin in whole blood: whole blood is applied directly, without removal of blood cells and without removal of other constituents of blood, onto an electrode to which an oligopeptide derivative of the formula (I) or a salt thereof and possible water-soluble additions are adhering.
The substrate of the determination method of the invention, namely an oligopeptide derivative of the formula (I), consists of an oligopeptide residue to which the residue of a water-soluble aromatic or heteroaromatic amine is bonded. These residues of aromatic or heteroaromatic amines have additional functional groups such as, for example, hydroxyl groups or halogen atoms, which advantageously contribute to a lower electrochemical potential and crucially determine the solubility of the substrate in aqueous medium.
The oligopeptide derivatives of the formula (I) and salts thereof can be prepared according to the invention by using methods customary in peptide chemistry (general methods of M. Bodanszky xe2x80x9cThe Practice of Peptide Synthesisxe2x80x9d Springer Verlag, 2nd edition 1994) to link an amine of the formula Hxe2x80x94R4 in which R4 has the above meaning to the carboxyl group of arginine, whose amino group is protected or already has the appropriately protected residue of the peptide part of the desired product or a part thereof, and whose arginine group is protected, and, if necessary, the peptide part of the desired product is assembled completely, after which, if desired, the remaining protective group(s) is/are cleaved off and, if desired, a free amino group is acylated and/or, if desired, a resulting oligopeptide derivative of the above formula (I) is converted into an acid addition salt and/or a resulting acid addition salt of such an oligopeptide derivative is converted into a free oligopeptide derivative or into another salt.
The procedure can be such, for example, that the electroactive amino group is attached to the carboxyl group of the C-terminal arginine, its amino group being protected by a protective group, for example a benzyloxycarbonyl or tert-butyloxycarbonyl group, and the guanidino group of arginine being protected by protonization, for example with HCl or p-toluenesulfonic acid. The C-terminal electroactive amino group likewise serves as protective group during the stepwise assembly of the peptide chain. The other protective groups can be cleaved off selectively as required in order to attach the other amino acid residues, until the desired chain length is completely assembled. Finally, the remaining protective groups can be cleaved off completely without involving the electroactive amino group.
Oligopeptide derivatives with free N-terminal amino group can be acylated, for example with t-butylphenylsulfonyl chloride, tosyl chloride, acetyl chloride, butyl chloride, octanoyl chloride, benzoyl chloride, p-methylbenzoyl chloride, 2-chlorobenzoyl chloride, methylsulfonyl chloride, n-butylsulfonyl chloride, t-butylsulfonyl chloride, isopropylsulfonyl chloride, phenylsulfonyl chloride, 1- or 2-naphthylsulfonyl chloride, (+)- or (xe2x88x92)-camphorsulfonyl chloride or with malonyl chloride.
Preferred salts of oligopeptide derivatives of the formula (I) are those in which the strongly basic guanidino group of arginine is stabilized by protonization with HCl, HBr, H2SO4 or H3PO4 or with formic, acetic, propionic, phthalic, citric, oxalic, tartaric, benzoic, lactic, trichloroacetic or trifluoroacetic acid, in particular with HCl, acetic acid or trifluoroacetic acid.
In the determination method of the invention, the enzyme to be determined cleaves the bond between the carboxy-terminal amino acid arginine and the water-soluble aromatic or heterocyclic amine. The measurement method comprises electrochemical determination of the amount of liberated amine (xe2x80x9celectroactive speciesxe2x80x9d). This electrochemcial determination method is suitable for determining proteases of the blood coagulation systems, in particular thrombin.
The substrate of this determination method consists, for example, of tosyl-glycyl-prolyl-arginyl-3-chloro-4-hydroxyanilide monoacetate salt, in which case the following enzymatic reaction occurs:
Substrate: Tos-Gly-Pro-Arg 3-chloro-4-hydroxyanilidexc3x97AcOH+enzyme↓thrombin
Product: Tos-Gly-Pro-Arg-OH+4-amino-2-chlorophenol
The particular advantage of the determination method of the invention is that the substrates are dissolved homogeneously in any aqueous reaction medium without addition of solubilizers such as, for example, DMSO. It is therefore possible with this electrochemical determination method to determine the enzyme concentration in the reaction medium with high measurement accuracy.